Bioactive whey protein hydrolysate

ABSTRACT

The present invention relates to a process of preparing a whey protein hydrolysate from a WPI substrate having improved flavour, functionality and ACE-I inhibiting properties.

TECHNICAL FIELD

[0001] This invention relates to a process for producing improvedhydrolysed whey protein products which are substantially free of bitterflavours and which contain bioactive peptides. The products of theprocess have high digestibility and good organoleptic properties. Theproducts have a bland taste and are free of soapy or brothy flavours.The improved hydrolysed whey protein products are useful sources ofbioactive peptides for incorporation into functional foods.

BACKGROUND ART

[0002] A number of food ingredients and foodstuffs have been producedfrom the hydrolysis of a protein source such as the milk proteins,casein and whey proteins.

[0003] Hydrolysed protein foodstuffs may have advantages overnon-hydrolysed protein foodstuffs in a number of areas of health care.For example, it is known that enzymatically hydrolysed proteins are lessallergenic. They are also more rapidly digested and absorbed than wholeproteins. Foodstuffs containing hydrolysed proteins are also useful inthe alimentation of hospital patients with digestive diseases forexample.

[0004] Hydrolysis of whey proteins and caseins is known to releasebioactive peptides that can exhibit a number of physiological effects(Maubois et al, 1991; EP 4745506). A number of publications describesuch bioactive peptides, for example, ACE inhibiting peptides which haveantihypertensive properties have been released through an enzymatictreatment of β-lactoglobulin and whey protein concentrates (Mullally etal, 1997). ACE inhibitory peptides are also found in sour milk and inhydrolysates of α, and β casein (JP 4282400; Nakamura et al 1994,Yamamoto 1997).

[0005] EP 4745506 discloses the hydrolysis of the milk proteinlactoferrin in whey to release lactoferrin which acts as anantimicrobial agent useful for treating diarrhoea, athlete's foot, eyeinfections, mastitis etc in humans and animals.

[0006] However, the hydrolysis of most food proteins, especially thehydrolysis of whey and casein containing products, is known to generatebitterness. This causes palatability problems particularly whenattempting to formulate orally ingestible products incorporating milkprotein hydrolysates as a source of bioactive peptides.

[0007] In the field of protein hydrolysis one or both of two approachesare commonly used for controlling or removing bitterness in proteinhydrolysates to increase palatability of the products.

[0008] The extensive hydrolysis of the protein substrate is known toreduce bitterness in milk protein hydrolysates (EP 065663; EP 0117047;U.S. Pat. No. 3,970,520). Less bitter products are produced relativelyeasily and cheaply in this way. However, extensive hydrolysis reducesthe chain lengths of all peptides, including the bioactive peptides ofinterest. Extensive hydrolysis of the protein substrate destroys thefunctional and biological activity of the peptide of interest. Inaddition soapy and brothy off-flavours often develop, with theconsequence that the palatability of the final product remains poorcompared to the original bland tasting protein substrate. A finaldisadvantage is that for some hydrolysates the bitterness is onlypartially removed (Roy 1992 and 1997).

[0009] A second common method for the control of bitterness in proteinhydrolysates is to use debittering enzymes, in particular those sourcedfrom Aspergillus oryzae.

[0010] “Bitterness” generation in protein hydrolysis is thought to bedue to the presence of large hydrophobic ‘bitter’ peptides. Debitteringeyes selectively hydrolyse bitter peptides present in the proteinhydrolysates. A worker skilled in the art can—by the judicious selectionof debittering enzymes and the conditions of treatment—effectivelydebitter milk protein hydrolysates leaving intact the particularbioactive peptides of interest. However, use of debittering enzymesmakes the process more expensive, and preservation of some of thebioactive peptide is not easily or successfully achieved. A furtherdisadvantage is that debittering enzymes treatments have a tendency torelease free amino acids into the final product and, as a consequence,the hydrolysates develop unpleasant brothy or soapy flavours (Roy 1992and 1997).

[0011] The various methods of debittering the protein hydrolysatesresult in additional process steps and add costs to the manufacture ofthe final product. In addition the final product also becomesoverbalanced in its supply of free amino acids.

[0012] It would be most advantageous if a process for hydrolysingprotein could be developed which releases bioactive peptides of interestand which limits the formation of bitter peptides and free amino acids,thereby allowing the original bland taste of the milk protein substratesto be retained.

[0013] Some bioactive peptides—in particular the antihypertensivepeptides—are relatively stable during protein hydrolysis and arereleased very early during the hydrolysis of the milk protein substrateas shown in FIG. 1.

[0014] The bitter flavours of milk protein hydrolysates can be improvedby adding sugars or by hydrolysing natural sugars, such as lactose,already present in the milk protein substrate (Bernal and Jelen, 1989).For example sour wheys and cheese wheys are made more palatable whenthey have been sweetened by β-galactosidease and lactase hydrolysis oflactose (FR 2309154; U.S. Pat. No. 4,358,464; JP 8056568).

[0015] In order to achieve a high flavour acceptability for a hydrolysedprotein product which contains bioactive peptides, precise control ofhydrolysis is required to prevent bitterness occurring.

[0016] A common method of termination of hydrolysis is by deactivationof the enzymes, usually by thermal deactivation at high temperatures,typically >90-100° C. for an extended period of time. However, thismethod cannot be used to stop the hydrolysis of whey proteins as anyintact unhydrolysed whey proteins remaining in the mixture woulddenature and precipitate making the final product less soluble and lessacceptable for the use as a food ingredient.

[0017] Such a problem was overcome in. WO 99/65326 which discloses aprocess of mid hydrolysis of sweet whey or sweet whey proteinconcentrate (WPC) to produce hydrolysates containing bioactive peptideshaving one or more of the following properties:

[0018] antihypertensive ACE-I activity

[0019] bifidus growth promoting activity

[0020] non-gluey, non-bitter flavour

[0021] pleasant to slightly sweet taste

[0022] good organoleptic properties.

[0023] The present invention uses a different whey protein-containingsubstrate to that used in WO 99/65326 although a similar hydrolysingprocess is used. Surprisingly, the use of this different substrateresults in a hydrolysate which shows dramatic improvements in the abovementioned properties of the whey hydrolysates, particularly in theantihypertensive ACE-I activity, flavour and functionality of theproduct.

[0024] It is broadly to the process of hydrolysing a different wheyprotein containing substrate and the novel hydrolysate produced by thisprocess that the present invention is directed.

SUMMARY OF THE INVENTION

[0025] Accordingly the invention may be said broadly to consist in aprocess for preparing an improved whey protein hydrolysate containingbioactive peptides comprising hydrolysing a whey protein isolate (WPI)with one or more enzymes characterised in that:

[0026] i) the enzyme is a heat labile protease;

[0027] ii) the hydrolysis is conducted at a temperature of between about30° C. and 65° C. at a pH of about 3.5 to about 9.0 when said enzyme isa neutral protease, at a pH of about 2.5 to about 6.0 where said enzymeis an acid protease; and at a pH of about 5.0 to about 10.0, where saidenzyme is an alkaline protease;

[0028] iii) the hydrolysis is terminated when a degree of hydrolysis ofno greater than about 10% has been reached;

[0029] iv) the hydrolysis is terminated by deactivating said one or moreenzymes; and

[0030] v) the conditions for said step iv) are sufficiently mild toavoid substantial denaturation of peptides or residual proteins in saidhydrolysate;

[0031] wherein the product produced by thee process is highly soluble.

[0032] By WPI is meant a whey protein isolate produced by any methodknown in the art. Preferably the WPI is produced by a method of ionexchange from a whey protein concentrate, such as a cheese, acid, orlactic whey protein concentrate, as would be appreciated by a personskilled in the art.

[0033] By heat labile is meant hat the enzyme is susceptible toirreversible deactivation at relatively moderate temperatures as wouldbe appreciated by a person skilled in the art.

[0034] Preferably the enzyme is selected from the group consisting ofProtease P6, Protease A, Protease M, Peptidase, Neutrase, Validaise, AFP2000, and any other heat labile protease.

[0035] The enzyme hydrolysis step may be carried out under conditionswhich are suitable for the particular enzyme used as would be understoodby a person skilled in the art.

[0036] The whey protein isolate (WPI) may be hydrolysed at aconcentration in the range of from about 5-35% solids and the enzyme orenzyme mixtures may be added to give an enzyme to substrate ratiobetween about 0.01% and about 3% w/w total solids, preferably betweenabout 0.01% and about 1.0% w/w total solids.

[0037] WPI treated with acid proteases may be hydrolysed at a pH ofbetween about 2.5 and about 6.0, preferably between a pH of about 3.0and about 5.0.

[0038] WPI treated with neutral proteases may be hydrolysed at a pH ofbetween about 3.5 and about 9.0, preferably between a pH of about 6.0and about 8.0.

[0039] WPI treated with alkaline proteases may be hydrolysed at a pHrange of between about 5.0 and about 10.0, preferably between a pH ofabout 6.0 and about 8.0.

[0040] The protein hydrolysis may be carried out at a temperature rangeof from between about 30-65° C., preferably from about 5060° C.

[0041] In one embodiment the one or more enzymes used to selectivelyhydrolyse the WPI may be immobilised on an inert support during saidhydrolysis step ii) wherein said inert support; is Roelun Eupergit,carrageenan particles, chitosan particles or any other suitable inertsupport material. This enzyme system may then be used in a stirred tankor fixed bed reactor or on a membrane or hollow fiber reactor to carryout the hydrolysis reaction.

[0042] The enzyme(s) used for hydrolysis may be cross linked to the saidinert support prior to the hydrolysis reaction.

[0043] The hydrolysate of the present invention is referred to as a“mild” hydrolysate whereby the degree of hydrolysis, i.e. the percentageof peptide bonds cleaved by enzymic action is less than about 10%. Thus,although the hydrolysate may still contain large peptide chains that maybe slightly denatured, the final hydrolysate product is highly soluble.

[0044] The degree of hydrolysis of the WPI substrate is preferably fromabout 3% to about 10%, most preferably from about 3% to about 5% beforethe hydrolysis is terminated.

[0045] Hydrolysis is determined by the enzyme deactivate step iv).Preferably, enzyme deactivation comprises heat deactivation.

[0046] The heat deactivation may comprise heating said hydrolysate forup to about ten seconds to a temperature of up to about 100° C.

[0047] When the hydrolysis is conducted at a temperature of below 65°C., the heat deactivating step is conducted at about 65° C. to about 70°C. for from about 10 seconds to about 15 minutes.

[0048] When the hydrolysis is conducted at a temperature of below 60°C., the heat deactivating step is conducted at about 60° to about 65° C.for from about 10 seconds up to about 30 minutes.

[0049] Alternatively, the enzyme deactivating step iv) comprisesaltering the pH of said whey protein-containing substrate to a pH atwhich said protease is not active.

[0050] According to one option, depending on the enzyme(s) used, theenzyme or enzyme mixture may also be deactivated by the evaporation anddrying procedures.

[0051] According to another option the enzyme or enzyme mixture may alsobe deactivated with or without a prior pH change.

[0052] Alternatively, the enzymes may be deactivated by simply removingthem from the reaction mixture. For example, when the one or moreenzymes used to selectively hydrolyse the WPI are immobilised on aninert support, such as by cross-linking to said inert support prior tothe hydrolysis reaction, they may be subsequently separated out of thehydrolysis reaction by membrane filtration in order to achievedeactivation.

[0053] Alternatively, the enzyme(s) may be separated out of thehydrolysis mixture with the use of an ultrafiltration membrane with anominal molecular weight cutoff in the range of about 10-500 kDa,preferably about 10-200 kDa, once hydrolysis is complete.

[0054] In a preferred embodiment, termination of the hydrolysis isachieved by deactivating the one or more whey protein hydrolysis enzymesby firstly changing the pH of the reaction mixture to a pH in which theenzyme(s) is either inactive or less active, and/or heating the reactionmixture to a comparatively mild temperature using a heat exchanger todenature the enzyme but not the intact whey proteins in the substrate. Asuitable temperature range which would denature the enzymes is in theorder of about 55-70° C., preferably about 65° C.

[0055] After hydrolysis and optional deactivation or removal of enzymes,the hydrolysate may optionally be subjected to reverse osmosis underconditions whereby salt and water are removed from the hydrolysate. Thepurified desalted hydrolysate comprising whey proteins, polypeptides andbioactive peptides is then recovered.

[0056] Optionally the hydrolysed WPI containing the bioactive peptidefraction can be purified with a UF membrane of about 5-200 kDa cut off,preferably about 10-50 kDa cut off. The bioactive peptides, and otherpeptides are recovered in the permeate.

[0057] According to another option ion exchange or hydrophobicadsorption or hydrophobic interaction chromatography or combinations ofthese processes may be used to recover the hydrolysed bioactive fractionfrom the hydrolysates in an enriched form.

[0058] In another embodiment the invention consists in a whey proteinhydrolysate containing one or more bioactive peptides selected from thegroup consisting of SAP (SEQ ID NO: 1), MKG (SEQ ID NO: 2), ALPMH (SEQID NO: 3), LIVTQ (SEQ ID NO: 4), VSLPEW (SEQ ID NO: 5), INYWL (SEQ IDNO: 6), LKPTPEGDLEIL (SEQ ID NO: 7) and LKGYGGVSLPEW (SEQ ID NO: 8).

[0059] Preferably the whey protein hydrolysate comprises at least onebioactive peptide selected from the group consisting of LIVTQ (SEQ IDNO: 1), MKG (SEQ ID NO: 2) and ALPMH (SEQ ID NO: 3), in combination withat least one bioactive peptide selected from the group comprising SAP(SEQ ID NO: 4), VSLPEW (SEQ ID NO: 5), INYWL (SEQ ID NO: 6),LKPTPEGDLEIL (SEQ ID NO: 7) and LKGYGGVSLPEW (SEQ ID NO: 8).

[0060] According to a further aspect the present invention provides apharmaceutical composition comprising one or more of the bioactivepeptides produced in the process of the invention together with apharmaceutically acceptable carrier. Preferably said pharmaceuticalcomposition comprising at least the bioactive peptide MKG (SEQ ID NO: 2)together with a pharmaceutical acceptable carrier.

[0061] According to a still further aspect, the present inventionprovides a method of treating or preventing hypertension in a mammalcomprising administering an effective amount of a bioactive peptideproduced by hydrolysing WPI according to the process of the invention toa mammal in need thereof.

[0062] According to a still flier aspect, the present invention providesa use of one or more bioactive peptides produced by the process of theinvention in the manufacture of a medicament for treating or preventinghypertension in a patient in need of such treatment. Preferably saidbioactive peptide is MKG (SEQ ID NO: 2).

[0063] The present invention further provides a non-bitter, highlysoluble WPI hydrolysate product prepared by the process of theinvention, wherein the degree of hydrolysis of WPI is about 3% to about10%. The mean particle size of whey proteins in the product may be lessthan about 30 microns, preferably less than about 3 microns.

[0064] In a further embodiment the invention provides a food productcontaining a WPI hydrolysate of the invention.

[0065] The invention further comprises a method of reducing systolicblood pressure in a subject, wherein said method comprises administeringan effective amount of the novel WPI hydrolysate or food productcontaining said hydrolysate of the invention to a subject in needthereof.

[0066] The invention further comprises one or more peptides selectedfrom the group comprising SAP (SEQ ID NO: 1), MKG (SEQ ID NO: 2), ALPMH(SEQ ID NO: 3), LIVTQ (SEQ ID NO: 4), VSLPEW (SEQ ID NO: 5), INYWL (SEQID NO: 6), LKPTPEGDLEIL (SEQ ID NO: 7) and LKGYGGVSLPEW (SEQ ID NO: 8).

[0067] The invention further comprises at least one bioactive peptideselected from the group consisting of LIVTQ (SEQ ID NO: 1), MKG (SEQ IDNO: 2) and ALPMH (SEQ ID NO: 3), in combination with at least onebioactive peptide selected from the group comprising SAP (SEQ ID NO: 4),VSLPEW (SEQ ID NO: 5), INYWL (SEQ ID NO: 6), LKPTPEGDLEIL (SEQ ID NO: 7)and LKGYGGVSLPEW: (SEQ ID NO: 8).

[0068] The hydrolysed WPI product of the invention has one or more ofthe following features;

[0069] antihypertensive ACE-I activity

[0070] probiotic growth promoting activity

[0071] non-gluey, non-bitter flavour

[0072] pleasant to slightly sweet taste

[0073] good organoleptic properties

[0074] high solubility

[0075] very good foaming properties

[0076] very good gelling properties

[0077] improved heat stability

[0078] The application of the mild hydrolysis technology to thesubstrate of whey protein isolates (WPI) revealed some dramaticimprovements of the final product in comparison to sweet whey proteinconcentrate (WPC) as a whey-protein containing substrate as disclosed inWO 99/65326 as follows:

[0079] Solubility

[0080] Although the WPI still denatures slightly on the heat conditionsto stop the protease reaction, it does not produce any insolublematerial. The solubility remains around 96% which is greater than thecorresponding WPC hydrolysate.

[0081] Heat Stability

[0082] The hydrolysed WPI was significantly more heat stable than thehydrolysed WPC. After 120° C. for 10 min @ 5% TS the solubility remains95%.

[0083] Appearance

[0084] Proper selection of reaction conditions can determine whether thefinal hydrolysate will look white or clear in solution. Example 1,below, produces an opaque product, whilst Examples 2 and 3 (below)result in clear product in solution at neutral pH. Hydrolysates of WPCwere all substantially white in appearance.

[0085] Foaming Ability and Stability

[0086] The hydrolysed WPI shows about double the foaming ability andabout four times the foam stability of non-hydrolysed WPI. This makesthe product very suitable as an ingredient for yoghurt and dessertapplications. The hydrolysed WPI also showed improved foaming abilityand stability compared to a WPC hydrolysate.

[0087] Gel Strength

[0088] The mildly hydrolysed WPI shows a markedly increased gel strengthcompared to non-hydrolysed WPI. This makes the product very suitable asan ingredient for yoghurt and dessert applications. The hydrolysed WPIalso showed improved gel strength compared to a WPC hydrolysate.

[0089] Flavour

[0090] The hydrolysed WPI shows significantly less bitterness comparedto mildly hydrolysed WPC products. The bitterness shows no tendency toincrease over time of hydrolysis, making the control of the process mucheasier in comparison to WPC hydrolysis.

[0091] ACE-I Activity In Vitro

[0092] The hydrolysed WPI shows about double the ACE-I activity in vitroof mildly hydrolysed WPC, given comparable; reaction conditions andenzyme addition.

[0093] Acceleration of Probiotic Fermentation

[0094] The hydrolysed WPI shows an acceleration in the rate of probioticyoghurt fermentation time of about 40% at an addition rate of 1.5%.

[0095] The invention consists in the foregoing and also envisagesconstructions of which the following gives examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] The present invention will now be described with reference to theaccompanying drawings in which:

[0097]FIG. 1 is a plot of bitterness and bioactivity on the ordinantagainst the degree of hydrolysis on the abscissa. The ‘opportunitywindow’ of obtaining a product according to the present inventioncontaining bioactive peptides and having acceptable flavour before thehydrolysis reaction produces bitter peptides is between the lines x₁,and x₂.

[0098]FIG. 2 shows the Acute Short Term Effect of Systolic BloodPressure of dosing mature spontaneously hypertensive rates (SHRs) with6.0 g WPC hydrolysate/kg body weight.

[0099]FIG. 3 shows the Acute Short Term Effect of Systolic BloodPressure of dosing mature spontaneously hypertensive rates (SHRs) with3.6 g WPI hydrolysate/kg body weight.

[0100]FIG. 4 shows the Acute Short Term Effect of Systolic BloodPressure of dosing mature spontaneous hypertensive rates (SHRs) with 165mg peptide MKG (SEQ ID NO: 2)/kg body weight.

DETAILED DESCRIPTION OF THE INVENTION

[0101] As discussed above, the present invention provides a process forproducing a hydrolysed WPI product containing bioactive peptides,whereby the hydrolysis is carried out under a high degree of control toprevent undesirable flavours developing during hydrolysis (e.g. bitter,soapy and brothy). The hydrolysis is terminated within the “opportunitywindow”, i.e. before the emergence of substantial bitterness—as shown inFIG. 1—to provide hydrolysates having good organoleptic properties andmaximum bioactive peptides. In FIG. 1 the degree of hydrolysis isrepresented qualitatively on the x axis. The window of opportunity isbetween the points x₁ and x₂ which will vary depending on the enzymewhich is used. The optimum conditions sought are a maximum bioactivitywith an acceptable level of bitterness.

[0102] In particularly preferred embodiments of the process of theinvention, the enzyme which hydrolyses the WPI is heat labile and isselected from the group consisting of Protease P6, Protease A, ProteaseM, Peptidase, Neutrase, Validase and AFP 2000 (all as herein defined)and the hydrolysis of the WPI is terminated by heat treatment for ashort time at a high temperature (about 85-100° C. for about 1-10seconds). The applicants have surprisingly found that the above enzymes(1) are able to produce a whey protein hydrolysate containing a goodlevel of bioactive peptides, and (2) can be inactivated by a short time,high temperature treatment which causes only partial denaturation of thewhey proteins in the hydrolysate, and surprisingly improves theorganoleptic properties of the whey proteins, in terms of providing aproduct which is substantially white or clear in appearance.

[0103] The present invention is now exemplified by the followingexamples using ALACEN™ 895 or ALACEN™ 894 (whey protein isolates,commercially available from NZDB), the product specifications for whichare attached in Appendix I:

EXAMPLE 1

[0104] Pilot Plant Production of WPI Mild Hydrolysate

[0105] Whey protein isolate produced by cation ion exchange technology(ALACEN™ 895) with a protein content ≧90% w/w was reconstituted at 20%total solids in water (50° C.) Reconstituted ALACEN™ 895 was transferredto a 150 L tank at 50° C. Water (50° C.) was added to the tank to makefinal total solids of 4%. The solution was stirred and Neutrase (E:S0.9%) was added.

[0106] Two hours after enzyme addition the first hydrolysate was pumpedthrough the UHT plant. Enzyme inactivation was achieved using directsteam injection to heat the hydrolysate to 88° C. and the hydrolysatewas held it at this temperature for 1.5 seconds.

[0107] The hydrolysate was flash cooled and passed through shell andtube heat exchanges to cool to ambient temperature.

[0108] The hydrolysate was subsequently evaporated and dried.

[0109] A hydrolysate made following the process of Example 1 had thefollowing features:

[0110] Solubility: 95%

[0111] Heat stability: 120° C. for 10 min @5% TS solubility 95%

[0112] ACE-I in vitro activity: 289 mg/L IC50

[0113] Foaming: Markedly increased over non-hydrolysed WPI

[0114] Flavour: Markedly improved over WPC based mild hydrolysates

[0115] Appearance: opaque white, particle size ˜0.1 μm

[0116] The solubility, heat stability, foaming, and appearance of theWPI hydrolysate were measured by standard methods familiar to thoseskilled in the art. The solubility of 5% total solids (TS) solution wasdetermined by centrifugation at 3000 g for 10 mins (at roomtemperature). The heat stability of 5% TS solutions was determined byheating to 120° C. for 10 mins, quick cooling, then centrifugation at700 g for 10 mins. The TS contained in the supernatant and the originalsolution was determined. The solubility was defined as theTS(supernatant)/TS(original solution) Foaming of 10% TS solutions at pH7.0 was determined by whisking with a Hobart mixer (Model N-50G, HobartCorporation) for 15 mins. The percentage overrun was used to compare thesample as would be understood by a person skill in the art. Theappearance of 5% TS was determined by visual observation and theparticle size was measured using a Malvern MasterSizer (Model MSEOOSM,Malvern Instruments Ltd).

[0117] ACE-I activity (in vitro) in the dried product was determinedusing FAPGG as a substrate (Product 305-10 ex Sigma ChemicalCorporation, St Louis, Mo., USA) according of the method of D W Cushman& H S Cheung (1971). ACE-I activities are expressed as the amount ofmaterial (mg/L) needed to reduce the activity of the ACE-I enzyme by50%.

[0118] The flavour was assessed subjectively with reference to thebitterness and astringency of the hydrolysates, in particular, thesensory profile was assessed by a formal sensory panel. 5% w/w sampleswere tasted at 24° C. using multi-dimensional scaling. Samples wereevaluated and scored on a 150 mm anchored line (absent (0) to intense(150)).

EXAMPLE 2

[0119] The hydrolysis reaction was repeated as outlined above forExample 1(10% total solids, E:S 0.9%, 150 L). After inactivation thehydrolysate was immediately evaporated and dried

[0120] A hydrolysate made by this method had the following featureswhich were measured as disclosed above for Example 1:

[0121] Solubility 97%

[0122] Heat stability: 120° C. for 10 min @5% TS solubility 96%

[0123] ACE-I in vitro activity: 503 mg/L IC50

[0124] Foaming: Markedly increased over non-hydrolysed WPI

[0125] Gelling: Markedly increased over non-hydrolysed WPI

[0126] Flavour: Markedly improved over WPC based mild hydrolyses

[0127] Appearance: clear yellowish

EXAMPLE 3

[0128] The hydrolysis reaction of Example 1 was repeated as outlinedabove (4% total solids, E:S 0.9%, 150 L). Four hours after enzymeaddition the first hydrolysate was pumped through the UHT plant usingthe same conditions as in Example 1.

[0129] A hydrolysate made by is method had the following features whichwere measured as disclosed above for Example 1:

[0130] ACE-I in vitro activity: 230 mg/L IC50

[0131] Foaming: Markedly increased over non-hydrolysed WPI

[0132] Gelling: Markedly increased over non-hydrolysed WPI

[0133] Flavour: Markedly improved over WPC based mild hydrolysates

[0134] Appearance: clear yellowish

EXAMPLE 4

[0135] A 2% solution of ALACEN™ 89.4(micro-filtered WPI) was altered topH 3.0 before undergoing ultrafiltration at 10° C. with a 3,000 Daltonnominal molecular weight cutoff membrane (CDUF001LB, MilliporeCorporation, Bedford). The pH of the retentate was altered to 7.0 anddiluted to 2% total solids before ultrafiltration at 10° C. with thesame membrane used previously. The total solids of the retentate wereadjusted to 5.0% before being hydrolysed with E:S 0.9% w/w Neutrase(Novo Nordisk, Denmark) at: 50° C. After 4 h the sample was inactivatedat 88° C. for 3 seconds and subsequently freeze-dried. The ACE-Iactivity was determined to be 227 mg/L.

EXAMPLE 5

[0136] Mildly hydrolysed WPI from Example 3 was shown to promote thegrowth of a probiotic microorganism when added to half fat milk at anaddition rate of 1.5%. The milk was heat treated @ 90° C. for 10minutes. After cooling to 37° C. the milk was inoculated with 0.1% S.thermophilus and 2% L. rhamnosus HN001(DR20™). The fermentation time ofthe control was 20 h to reach the required pH of 4.4. In contrast, theWPI hydrolysate addition reduced the fermentation time to 13 h.

EXAMPLE 6

[0137] Identification of ACE-Inhibitor Peptides in WPI Hydrolysates

[0138] The objective of this example was to isolate and identifyACE-inhibitor peptides present in the WPI hydrolysates

[0139] Methodology

[0140] The starting material for ACE-I peptide isolation was a UFpermeate (10 kDa) obtained from the original hydrolysate of Example 1;

[0141] Peptides present in the hydrolysate were separated usingreverse-phase HPLC;

[0142] Purified peptides were assayed individually for ACE-inhibitoractivity as described in Example 1,

[0143] The amino acid sequence of each active peptide was identified bya combination of mass spectrometry and N-terminal sequence analysis;

[0144] The origin of the active peptides was determined by comparingtheir sequences with the known sequences of the milk proteins.

[0145] The peptides, their origins, activities known similarities areset out in Table 1 below: TABLE 1 ACE-inhibitor peptides isolated from aWPI hydrolysate ¹Activity Peptide ACE-inhibitor sequence Origin (IC₅₀) 1SAP (SEQ ID NO: 1) β-LG(36-38) 22 2 ²MKG (SEQ ID NO: 2) β-LG(7-9) 23 3²ALPMH (SEQ ID NO: 3) β-LG(142-146) 23 4 ²LIVTQ (SEQ ID NO: 4) β-LG(1-5)19 5 VSLPEW (SEQ ID NO: 5) α-LA(21-26)  8 6 ³INYWL (SEQ ID NO: 6)³LKPTPEGDLEIL α-LA(101-105) {close oversize brace} 11 (SEQ ID NO: 7)β-LG(46-57) 7 LKGYGGVSLPEW α-LA(15-26)  7 (SEQ ID NO: 8)

EXAMPLE 7

[0146] Sensory Comparison of Mildly Hydrolysed WPI Compared to MildlyHydrolysed WPC and Unhydrolysed WPI

[0147] WPI was hydolysed as set out above in Example 1.

[0148] WPC was hydrolysed as set out in Example 1 of WO 99/65326.(ALACEN™ 392, 10%, hydrolysed and inactivated after 2 hours).

[0149] ALATAL™ 819 is a commercial product of the New Zealand DairyBoard; the summary of the product specification of which is attached inAppendix I.

[0150] A taste panel scored the bitterness and astringency of theproducts scored on a 150 mm anchored line (absent (0) to intense (150)).

[0151] Degree of Hydrolysis was determined using the ModifiedO-phthaldialdehyde (MOPA) method as described by Frister et al. (1988).TABLE 2 Mean score (/150) for WPI and hydrolysate samples evaluated by aformal sensory panel, degree of hydrolysis, and ACE-I activity ofstandard WPI and hydrolysed whey protein. Degree of ACE-I SensoryEvaluation Hydrolysis activity Product tested Bitter Astringent (%)(mg/L) ALACEN ™ 895 (WPI) 36.2 24 0 ˜2000 Mildly hydrolysed WPC 78 67.14.5 440 Mildly hydrolysed WPI 55.1 41.6 4.3 230 ALATAL ™ 819 93.1 53.310 —

[0152] Both mildly hydrolysed WPI, and mildly hydrolysed WPC were morebitter and astringent than the non-hydrolysed WPI product as expected.However, these products were significantly less bitter and astringentthan a more vigorously hydrolysed ALATAL∩ 819 (a product of the NewZealand Dairy Board, NZ).

[0153] Mildly hydrolysed WPI products gave more acceptable flavouredproducts and had significantly higher ACE-I activity, co pared to themildly hydrolysed WPC (described in WO 99/65326). The results alsoshowed that the mildly hydrolysed WPI was significantly less bitter andastringent than the equivalent WPC product, although the degree ofhydrolysis for both was similar. As well as having a more acceptableflavour profile, the ACE-I activity of the mildly hydrolysed WPI wasalmost twice that of the mildly hydrolysed WPC.

[0154] Although further hydrolysis of WPC may result in an increasedACE-I activity, a significant decrease in the flavour profile comparedto the mildly hydrolysed WPI is observed, compromising the flavourprofile due to increased bitterness and astringency.

EXAMPLE 8

[0155] Blood Pressure Measurement

[0156] Blood pressure was measured using a tail cuff monitor and apurpose designed apparatus for measuring the blood pressure of smallanimals [IITC Inc, 239 Victory Blvd., Woodland Hills, Calif. 91367,USA]. Each time point was the average of 3-5 readings taken withinapproximately 5-10 minutes.

[0157] The Animal Strain

[0158] Spontaneously hypertensive rats (SHRs) were sourced from theAnimal Resource Centre, Western Australia.

[0159] Sample Dosage

[0160] The test substances were given to each rat on a per kg bodyweight basis unless otherwise stated. The individual dose for eachanimal was incorporated into a flavoured jelly, which was readilyconsumed by the animals.

[0161] The three samples evaluated for short tern blood pressureresponse were a mildly hydrolysed whey protein concentrate (FIG. 2), amildly hydrolysed whey protein isolate (see FIG. 3) and a synthesisedpeptide from. Table 1, namely MKG (SEQ ID NO: 2) (see FIG. 4). The WPCwas prepared according to the methodology set out in example 1 of WO99/65326.

[0162] Acute or short term effects of agents were determined by treatingadult SHRs with the agent and then studying their blood pressureresponses over the following hours. The study detects if an agent lowersblood pressure that is already elevated.

[0163] Results

[0164] Short term dosing of SHRs with 3.6 g/kg body weight of WPIhydrolysate (FIG. 3) resulted in a significant reduction in bloodpressure compared to controls rats (p=0.0378). The reduction in bloodpressure was similar to that achieved in rats dosed with 6.0 g/kg bodyweight WPC hydrolysate (FIG. 2). Thus, the WPI hydrolysate appears to bemore active than the comparative WPC hydrolysate so that approximately50% less of the WPI hydrolysate is required to give a similar decreasein blood pressure (systolic) in mature SHRs.

[0165] Short term dosing of SHRs with 165 mg/kg body weight peptide MKG(FIG. 4) showed a much greater reduction in blood pressure compared tocontrol rats (p=0.0021 after 4 hours; p<0.0001 after 8 hours) as well ascompared to the mild WPI hydrolysate and mild WPC hydrolysate.

CONCLUSIONS

[0166] The process of mildly hydrolysing WPI according to the presentinvention provides useful WPI hydrolysates which show significant:functional improvement over the WPC hydrolysates of WO 99/65326. Inparticular, the present WPI hydrolysates comprise more active peptideswith significantly less WPI hydrolysate required to decrease bloodpressure in vivo to the same degree as a WPC hydrolysate WPI can behydrolysed for longer without losing acceptable: flavor, characteristicsand whilst maintaining the increased ACE-I activity. Thus, ova heprocess of the present invention provides a bioactive whey proteinhydrolysate which is improved in flavour, ACE-I activity andfunctionality compared to that of WO 99/65326.

INDUSTRIAL APPLICATION

[0167] The process of the present invention is useful to produce thenovel whey protein hydrolysate which has surprising beneficialproperties and can be used as a food or medicine as ananti-hypertensive.

[0168] It will be appreciated that it is not intended to limit theinvention to the aforementioned examples only, many variations, such asmight readily occur to a person skilled in the art, being possible,without departing from the scope of the appended claims.

REFERENCES

[0169] Bernal V & Jelen P (1989). Effectiveness of lactose hydrolysis inCottage cheese whey for the development of whey drinks. Milchwissenchaft44: 222-225

[0170] FR 2309154, 30 Dec. 1976 Fromageries Bel La Vache Qui (From),France.

[0171] U.S. Pat. No. 3,970,520, 20 Jul. 1976, General Electric Co, USA.

[0172] EP0117047, 29 Aug. 1984, General Foods Corporation, USA.

[0173] Maubois J L, Léonil J, Trouvé R & Bouhallab S(1991) Les peptidesdu lait à activité physiologique III. Peptides du lait à effectcardiovasculaire: activités antithrombotique et antihypertensive. Lait,71, 249-255.

[0174] JP 4282400, 7 Oct. 1992, Calpis Shokuhin Kogyo KK, Japan.

[0175] EP065663, 1 Dec. 1982, Miles Laboratories Incorporated, USA.

[0176] JP 8056568, 17 Aug. 1994. Morinaga Mil Co Ltd Japan.

[0177] EP4745506, 11 Mar. 1992, Morinaga Milk Co Ltd, Japan.

[0178] Mullally M M, Meisel H & FitzGerald R J (1997) Identification ofa novel angiotensin-I-converting enzyme inhibitory peptide correspondingto a tryptic fragment of bovine β-lactoglobulin. Federation of EuropeanBiochemical Societies Letters, 402, 99-111.

[0179] Nakamura Y, Yamamoto N, Sakai K & Takano T (1994).Antihypertensive effect of the peptides derived from casein by anextracellular proteinase from Lactobacillus helveticus CP790. Journal ofDairy Science, 77, 917-922.

[0180] Roy G (1992). Bitterness: reduction: and inhibition. Trends inFood Science and Technology 3: 85-91

[0181] Roy G (1997). Modifying bitterness: Mechanism, ingredients andapplications. Technomic Publishers, Lancaster, UK.

[0182] U.S. Pat. No. 4,358,464, 9 Sep. 1982, Superior Dairy Company,USA.

[0183] Yamamoto N (1997). Antihypertensive peptides derived from foodproteins. Biopolymers 43: 129-134.

[0184] Frister H, Meisel H & Schelimme E (1988). OPA method modified byuse of N,N-dimethyl-2-mercaptoethylammoniumchloride as thiol component.Freesenius Z Anal Chem. 330, 631-633

[0185] Cushman D W & Cheung H S (1971). Spectrophotometric assay andproperties of the angotension converting enzyme in rabbit lung. BiochemPharmocol 20: 1637-1648.

[0186] WO 9965326, 23^(rd) Dec., 1991, New Zealand Dairy Board, NZ.

1 8 1 3 PRT Bos taurus 1 Ser Ala Pro 1 2 3 PRT Bos taurus 2 Met Lys Gly1 3 5 PRT Bos taurus 3 Ala Leu Pro Met His 1 5 4 5 PRT Bos taurus 4 LeuIle Val Thr Gln 1 5 5 6 PRT Bos taurus 5 Val Ser Leu Pro Glu Trp 1 5 6 5PRT Bos taurus 6 Ile Asn Tyr Trp Leu 1 5 7 12 PRT Bos taurus 7 Leu LysPro Thr Pro Glu Gly Asp Leu Glu Ile Leu 1 5 10 8 12 PRT Bos taurus 8 LeuLys Gly Tyr Gly Gly Val Ser Leu Pro Glu Trp 1 5 10

What we claim is:
 1. A process for preparing an improved whey proteinhydrolysate containing bioactive peptides comprising hydrolysing a wheyprotein isolate (WPI) with one or more enzymes characterised in that: i)the enzyme is a heat labile protease; ii) the hydrolysis is conducted ata temperature of between about 30° C. and 65° C. at a pH of about 3.5 toabout 9.0 when said enzyme is a neutral protease, at a pH of about 2.5to about 6.0 where said enzyme is an acid protease; and at a pH of about5.0 to about 10.0 where said enzyme is an alkaline protease; iii) thehydrolysis is terminated when a degree of hydrolysis of no greater thanabout 10% has been reached; iv) the hydrolysis is terminated bydeactivating said one or more enzymes; and v) the conditions for saidstep iv) are sufficiently mild to avoid substantial denaturation ofpeptides or residual proteins in said hydrolysate; wherein theproduction of the process is highly soluble.
 2. A process according toclaim 1, wherein the enzyme is selected from the group consisting ofProtease P6, Protease A, Protease M, Peptidase, Neutrase, Validase, AFP2000, and any other heat labile protease.
 3. A process as claimed inclaim 1 or 2, wherein said enzyme deactivating step iv) comprises heatdeactivation.
 4. A process as claimed in claim 3, wherein said heatdeactivation comprises heating said hydrolysate for up to ten seconds toa temperature up to about 100° C.
 5. A process as claimed in claim 3,wherein, when said hydrolysis is conducted at a temperature of belowabout 65° C., said heat deactivating step is conducted at about 65° C.to about 70° C. for from about 10 seconds to about 15 minutes.
 6. Aprocess as claimed in claim 3, wherein, when said hydrolysis isconducted at a temperature below about 60° C., said heat deactivatingstep is conducted at about 60°to about 65° C. for from about 10 secondsup to about 30 minutes.
 7. A process as claimed in claim 1 or claim 2,wherein said enzyme deactivating step comprises altering the pH of saidwhey protein-containing substrate to a pH at which said protease is notactive.
 8. A process as claimed in claim 7, wherein said enzymedeactivating step includes heat deactivation as claimed in any one ofclaims 3 to
 6. 9. A process as claimed in claim 1 or claim 2, whereinsaid enzyme deactivating step iv) comprises subjecting said hydrolysateto ultrafiltration with an ultrafiltration membrane having a nominalmolecular weight cut off in the range of about 10-500 kDa.
 10. A processas claimed in claim 9, wherein said ultrafiltration membrane has anominal molecular weight cut off in the range of about 10-200 kDa.
 11. Aprocess as claimed in any one of the preceding claims, wherein saidenzyme is immobilised on an inert support during said hydrolysis stepii).
 12. A process as claimed in claim 11, wherein said inert support isRoehm Eupergit, carrageenan particles, chitosan particles or any othersuitable inert support material.
 13. A process as claimed in anyone ofthe preceding claims, wherein the degree of hydrolysis is from about 3%to about 10%.
 14. A process as claimed in claim 13, wherein the degreeof hydrolysis is from about 3% to about 5%.
 15. A process as claimed inany one of the preceding claims, wherein the whey protein hydrolysate soproduced comprises one or more bioactive peptides selected from thegroup consisting of SAP (SEQ ID NO: 1), MKG (SEQ ID NO: 2), ALPMH (SEQID NO: 3), LIVTQ (SEQ ED NO: 4), VSLPEW (SEQ ID NO: 5), INYWL (SEQ IDNO: 6), LKPTPEGDLEIL (SEQ ID NO: 7) and LKGYGGVSLPEW (SEQ ID NO: 8). 16.A process as claimed in any one of the preceding claims, wherein thewhey protein hydrolysate so prepared comprises at least one bioactivepeptide selected from the group consisting of LIVTQ (SEQ ID NO: 1), MKG(SEQ ID NO: 2) and ALPMH (SEQ ID NO: 3), in combination with at leastone bioactive peptide selected from the group comprising SAP (SEQ ID NO:4) VSLPEW (SEQ ID NO: 5), INYWL (SEQ ID NO 6), LKPTPEGDLEIL (SEQ ID NO:7) and LKGYGGVSLPEW (SEQ ID NO: 8).
 17. A pharmaceutical compositioncomprising one or more of the bioactive peptides produced in the processof any one of claims 1 to 16 together with a pharmaceutically acceptablecarrier.
 18. A pharmaceutical composition as claimed in claim 17,comprising the bioactive peptide MKG (SEQ ID NO: 2) together with apharmaceutical acceptable carrier.
 19. A method of treating orpreventing hypertension in a mammal, said method comprisingadministering an effective amount of a bioactive peptide produced byhydrolysing WPI according to the process of any one of claims 1 to 16 toa mammal in need thereof.
 20. A use of one or more bioactive peptidesproduced by the process of any one of claims 1 to 16 in the manufactureof a medicament for treating or preventing hypertension in a patient inneed of such treatment.
 21. A use as claimed in claim 19, wherein saidbioactive peptide is MKG (SEQ ID NO: 2).
 22. A non-bitter, highlysoluble WPI hydrolysate product containing bioactive peptides, preparedby the process of any one of claims 1 to
 16. 23. A product as claimed inclaim 22, wherein the degree of hydrolysis of the WPI is about 3% toabout 5%.
 24. A product as claimed in claim 23, wherein the mainparticle size of whey proteins in the product is less than about 30microns.
 25. A product as claimed in claim 24, wherein the main particlesize is less than about 3 microns.
 26. A product as claimed in any oneof claims 22 to 25, which is substantially clear or white in solution.27. A product as claimed in any one of claims 22 to 26, wherein one ormore of said bioactive peptides is selected from the group consisting ofSAP (SEQ ID NO: 1), MKG (SEQ ID NO; 2), ALPMH (SEQ ID NO: 3), LIVTQ (SEQID NO: 4), VSLPEW (SEQ ID NO: 5), INYWL (SEQ ID NO: 6), LKPTPEGDLEIL(SEQ ID NO: 7) and LKGYGGVSLPEW (SEQ ID NO: 8).
 28. A product as claimedin any one of claims 22 to 26, comprising at least one bioactive peptideselected from the group consisting of LIVTQ (SEQ ID NO: 1), MKG (SEQ IDNO: 2) and ALPMH (SEQ ID NO: 3), in combination with at least onebioactive peptide selected from the group comprising SAP (SEQ ID NO: 4),VSLPEW (SEQ ID NO: 5), INYWL (SEQ ID NO: 6), LKPTPEGDLEIL (SEQ ID NO: 7)and LKGYGGVSLPEW (SEQ ID NO: 8).
 29. A food product containing a WPIhydrolysate product as claimed in any one of claims 22 to
 28. 30. Amethod of reducing systolic blood pressure in a subject comprisingadministering an effective amount of a WPI hydrolysate as claimed in anyone of claims 22 to 28 or food product containing said hydrolysate asclaimed in claim 29 to a patient in need thereof.
 31. A use of a productas claimed in any one of claims 22 to 28 in the manufacture of amedicament for treating or preventing hypertension in a patient in needthereof.
 32. A pharmaceutical composition comprising the product of anyone of claims 22 to 28 together with a pharmaceutically acceptablecarrier.
 33. Any one or any combination of two or more peptides selectedfrom the group comprising SAP (SEQ ID NO: 1), MKG (SEQ ID NO: 2), ALPMH(SEQ ID NO: 3), LIVTQ (SEQ ID NO: 4), VSLPEW (SEQ ED NO: 5), INYWL (SEQID NO: 6), LKPTPEGDLEIL (SEQ ID NO: 7) and LKGYGGVSLPEW (SEQ ID NO: 8).34. Any one bioactive peptide selected from the group consisting ofLIVTQ (SEQ ID NO: 1), MKG (SEQ ID NO: 2) arid ALPMH (SEQ ID NO: 3), incombination with at least one bioactive peptide selected from the groupcomprising SAP (SEQ ID NO: 4), VSLPEW (SEQ ID NO: 5), OWL (SEQ ID NO:6), LKPTPEGDLEIL (SEQ ID NO: 7) and LKGYGGVSLPEW (SEQ ID NO: 8).